Systemic mastocytosis (SM), a myeloproliferative disorder with variable clinical manifestations, is associated in most cases with the D816V activating mutation in KIT which is the receptor for stem cell factor (SCF). The identification of the KIT D816V mutation in patients with systemic mastocytosis has gained a major prognostic significance in the last several years, largely because of the availability of tyrosine kinase receptor inhibitors such as imatinib. However, imatinib is ineffective in patients carrying KIT D816V mutation. This has lead us to investigate variants of KIT and to examine the consequences of a activating germ line mutation in KIT to better understand how such mutations alter mast cell proliferation and function. KIT is known to have two major variants which differ by four amino acids (GNNK- or GNNK+) at the juxta-membrane region of the extracellular domain. We examined the premise that the expression pattern of these variants differ in systemic mastocytosis, and that transcripts containing the KIT D816V mutation segregate preferentially to one GNNK variant. A quantitative real-time PCR assay to assess GNNK- and GNNK+ transcripts from bone marrow mononuclear cells was developed. We found that the GNNK-/GNNK+ copy number ratio positively trended with % neoplastic mast cell involvement and KIT D816V containing transcripts displayed a significantly elevated GNNK-/GNNK+ copy number ratio. Relative expression of the GNNK- variant, but not the GNNK+ variant, correlated with increasing percent of neoplastic mast cell involvement. A mast cell transfection system revealed that the GNNK- isoform of wild type KIT was associated with increased granule formation, histamine content and growth. These data support the conclusion that neoplastic mast cells favor a GNNK- variant predominance, which in turn enhances the activating potential of the KIT D816V mutation and thus may influence therapeutic sensitivity in systemic mastocytosis. Mastocytosis associated with germline KIT activating mutations is exceedingly rare and thus there are few studies on the effect of activating mutations on the biology of mast cells that continue to exhibit an activating mutation in KIT over time in culture. We were fortunate to be able culture mast cells from a patient who presented with unique clinicopathologic features of mastocytosis which was associated with a de novo germline KIT K509I mutation. To investigate the impact of the germline KIT K509I mutation on human mast cell development and function, primary human mast cells derived from CD34+ peripheral blood progenitors were examined for growth, development, survival and IgE-mediated activation. In addition, a mast cell transduction system which stably expressed the KIT K509I mutation was established. KIT K509I biopsied mast cells were round, CD25(-) and well differentiated. KIT K509I progenitors, cultured in SCF, demonstrated a ten-fold expansion compared to progenitors from healthy subjects. Further, they developed into mature, hypergranular mast cells with enhanced antigen-mediated degranulation. A KIT K509I mast cell transduction system revealed the SCF-independent survival to be reliant on the preferential splicing of KIT at the adjacent exonic junction. We concluded that germline KIT mutations associated with mastocytosis drive a well-differentiated mast cell phenotype, distinct to that of somatic KIT D816V disease, whose oncogenic potential may be influenced by SCF and selective KIT splicing.